Gene expression, which encompasses a series of reactions from initial gene activation to final protein folding, is an inherently noisy process for any cell population under study. As each step in the process is subject to independent regulatory pressures, small between cell differences in the levels of these regulators can produce substantial transcriptional heterogeneity, which may then propagate into substantial functional diversity. It is therefore challenging to understand how complex multi-cellular tissues faithfully develop given the number of genes that must be coordinately expressed to establish cellular identity. Master regulatory transcription factors (TF) are the proposed solution to this teleological dilemma. These molecules have been shown to control cohorts of genes required for normal cell function, and achieving the appropriate level and stoichiometry between different TF appears to be critical for fate decisions during normal tissue development. Moreover, the deregulation in either the expression or function of these factors appears to play a substantial role in malignant transformation. TF have been extensively studied in hematopoiesis, the highly arborized differentiation network that robustly and dynamically produces a spectrum of functionally distinct blood cell populations responsible for hemostasis, gas exchange, and immune function. In order to achieve this complex cellular output, hematopoietic differentiation is postulated to occur as a series of nodal fate decisions in increasingly oligopotent stem and progenitor cell compartments (HSPC), each with distinct gene expression programs governed by TF. Understanding how HSPC achieve the appropriate dose and activity of these TF is therefore vital to our understanding of steady state blood differentiation and may expose novel therapeutic windows in hematological disease. Complicating these efforts, however, is the finding that HSPC are functionally and transcriptionally heterogeneous, which have limited the field's ability to uncover definitive regulation of TF based on ensemble measurements. This project is intended to quantify the origins of that heterogeneity with single molecule, quantitative techniques to uncover the regulation and expression of a master hematopoietic TF, PU.1. Our proposal is to (1) determine how PU.1 mRNA and protein production is dynamically changed during differentiation in single primary HSPC from mice by RNA FISH/IF and to (2) independently measure how a highly conserved cis regulatory element (URE) controls the rate, magnitude, and dynamics of PU.1 transcription. Our preliminary findings have indicated that not only is our experimental approach feasible, it has already revealed intriguing findings about PU.1 mRNA synthesis that were previously unknown. Using these tools and sophisticated analytical techniques, this proposal will provide the highest resolution, quantitative study to date of the regulation and activity of a master regulatory transcription factor in primary HSPC. We anticipate that our approach will provide novel and fundamental insight into the molecular paradigms regulating hematopoiesis and leukemogenesis.

Public Health Relevance

Hematopoiesis is a complex, hierarchical system of differentiation nodes that depend on the proper induction of transcriptional programs in a timely fashion in oligopotent stem and progenitor cells, and PU.1 is a transcription factors that plays a critical role in the maintenance of those transcriptional states. Understanding how hematopoietic stem cells regulate the synthesis of PU.1 is a critical question to the fields of hematology and leukemia, given the finding that perturbations in the level or activity of this factor precipitates pathological conditions such as acute myeloid leukemia. Our approach to answering this question is to use quantitative, single molecule imaging tools with analyses of gene expression noise to measure the transcriptional kinetics of the PU.1 locus, in the hopes of inferring the upstream regulatory pressures that shape PU.1 levels in primary cells.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
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Special Emphasis Panel (ZRG1)
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Gibbs, Kenneth D
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Albert Einstein College of Medicine, Inc
United States
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Wheat, Justin C; Steidl, Ulrich (2018) Linking histone methylation, transcription rates, and stem cell robustness. Haematologica 103:1093
Carvajal, Luis A; Neriah, Daniela Ben; Senecal, Adrien et al. (2018) Dual inhibition of MDMX and MDM2 as a therapeutic strategy in leukemia. Sci Transl Med 10:
Wheat, Justin C; Steidl, Ulrich (2017) ETO2-GLIS2: A Chimeric Transcription Factor Drives Leukemogenesis through a Neomorphic Transcription Network. Cancer Cell 31:307-308
Sato, Hanae; Wheat, Justin C; Steidl, Ulrich et al. (2016) DNMT3A and TET2 in the Pre-Leukemic Phase of Hematopoietic Disorders. Front Oncol 6:187